Rabies virus-specfic neutralizing human monoclonal antibodies and nucleic acids and related methods

ABSTRACT

Human monoclonal rabies virus neutralizing antibodies represent a safe and efficacious post-exposure prophylactic therapy for individuals exposed to a rabies virus. The nucleic acid and encoded amino acid sequences of the heavy and light chain immunoglobulins of human monoclonal rabies virus neutralizing antibodies, and their use, is described.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 based uponU.S. Provisional Application No. 60/204,518, filed May 16, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to the fields of molecular biologyand immunology and, more particularly, to the nucleic acid and aminoacid sequence of human monoclonal rabies virus-neutralizing antibodies.

BACKGROUND OF THE INVENTION

[0003] Rabies is an acute, neurological disease caused by infection ofthe central nervous system with rabies virus, a member of the Lyssavirusgenus of the family Rhabdoviridae. Of great historical significance dueto its antiquity and the horrific nature of the disease, rabies viruscontinues to be an important threat of human and veterinary infectionbecause of extensive reservoirs in diverse species of wildlife.Throughout much of the world, distinct variants of rabies virus areendemic in particular terrestrial animal species, with relatively littlein common between them. While several islands, including the UnitedKingdom, Australia, Japan, and numerous islands are free of terrestrialrabies, rabies and rabies-related viruses associated with bats haverecently been identified in the UK and Australia.

[0004] Rabies virus is characteristically bullet-shaped, envelopedparticle of, on average, 75 by 180 nanometers. The virion consists of asingle-stranded negative sense RNA genome and five structural proteins:the nucleoprotein (N) molecules, the phospho-protein (NS), thepolymerase (L), the matrix protein (M) and the viral glycoprotein (G).

[0005] The N and G proteins both bear antigenic determinants whichenable serotypic characterization of diverse rabies virus strains. Ndeterminants are highly conserved between different virus isolates andare therefore very useful targets for the immunohistological detectionof rabies virus infection using specific antibodies. On the other hand,antigenic determinants carried on the G-protein vary substantially amongthe rabies virus strains. Virus-neutralizing antibodies raised byvaccination with inactivated virus are directed against G. While it isclear that T cell responses to G, N, and NS, participate in immuneresponses to the virus under experimental conditions, assessment ofimmunity to rabies virus is generally limited to serology, particularlywith respect to virus-neutralizing antibodies.

[0006] In areas of the world where human rabies is still common, the dogis the major reservoir of the viruses that infect man. Where caninerabies has largely been eliminated by vaccination, foxes, coyotes,skunks, raccoons, bats, and a variety of other mammals harbor variantsof the virus. In many areas, wildlife reservoirs of virus continue toexpand. Moreover, rabies virus can be transmitted from a reservoirspecies to humans or other end stage hosts by animals not normallyassociated with rabies, such as cats, rabbits, etc.

[0007] Almost invariably fatal once clinical symptoms appear, rabies canbe averted by prompt treatment of an infected individual with acombination of passive and active immunization. Passive immunizationconsists of the administration of pre-formed rabies virus neutralizingantibodies obtained from pooled serum of rabies immune individuals(Human rabies-immune globulin; HRIG) or hyper-immunized horses (Equinerabies-immune globulin; ERIG). Both types of reagent present certainrisks to recipients including variable antigen specificity, and thuspotency, for different rabies virus isolates.

[0008] HRIG is prepared from pooled human sera, therefore there is thepossibility that HRIG preparations could be contaminated with known orunknown human pathogens. On the other hand, as a preparation of foreignantigen, ERIG has been associated with severe anaphylactic reactions.Mouse monoclonals specific for rabies virus have been contemplated foruse in post-exposure prophylaxis but, like ERIG, are antigenicallyforeign to humans. This may result in their rapid clearance from thehuman system, as well as the potential to cause an anaphylacticreaction.

[0009] To provide a better reagent, human monoclonal antibodies havebeen made by fusion of Epstein-Barr Virus (EBV)-transformed, rabiesvirus-specific human B cells with mouse-human heterohybrid donors. cDNAclones encoding the antibody heavy and light chains from these cellswere constructed such that the antibodies were expressed in heterologousexpression systems. These constructs allow rabies virus neutralizinghuman antibodies of defined specificity to be produced in a controlledsystem, purified away from possible deleterious contaminants. Thepresent invention relates to these monoclonal rabies virus neutralizinghuman antibodies, the nucleic acid sequences of their heavy and lightchains and the amino acid sequences of the encoded proteins. Alsoprovided in the present invention are methods of using the monoclonalantibodies as a therapeutically effective post-exposure prophylactictreatment of individuals exposed to rabies virus.

SUMMARY OF THE INVENTION

[0010] It is an object of the present invention to isolate nucleic acidmolecules having a heavy chain and a light chain nucleic acid sequenceencoding a heavy chain and a light chain amino acid sequence. The heavychain and light chain amino acid sequences are that of a monoclonalrabies virus neutralizing antibody that specifically binds to a rabiesvirus protein.

[0011] In one embodiment of the present invention that the isolatednucleic acid molecules that encode the monoclonal rabies virusneutralizing antibody are derived from cDNA sequences of the heavy chainSEQ. ID. NO: 1 and the light chain SEQ. ID. NO: 2.

[0012] It is an object of the present invention to provide an isolatedhuman monoclonal rabies virus neutralizing antibody that is encoded incDNA clones encoding the antibody heavy and light chains expressed inheterologous expression systems and purified away from deleteriouscontaminants. In one embodiment of the present invention the amino acidsequence of the isolated human monoclonal rabies virus neutralizingantibody is that of the SEQ. ID. NO: 3 and SEQ. ID. NO:4, respectively.

[0013] The present invention provides a fused gene encoding a chimericimmunoglobulin light chain. The chimeric light chain contains a firstDNA sequence encoding an immunogloublin light chain variable region of amonoclonal rabies virus neutralizing antibody produced by aheterhybridoma cell line; and a second DNA sequence encoding a humanlight chain constant region. It is a further object of the presentinvention to provide an expression vector to express this fused gene. Itis a further object to provide a host cell for the expression vector.

[0014] The present invention provides a fused gene encoding a chimericimmunoglobulin heavy chain. The chimeric heavy chain contains a firstDNA sequence encoding an immunogloublin heavy chain variable region of amonoclonal rabies virus neutralizing antibody produced by aheterhybridoma cell line; and a second DNA sequence encoding a humanheavy chain constant region. It is a further object of the presentinvention to provide an expression vector to express this fused gene. Itis a further object to provide a host cell for the expression vector.

[0015] It is another object of the present invention to provide anisolated monoclonal rabies virus neutralizing antibody derived from thefused gene encoding a chimeric immunoglobulin light chain and the fusedgene encoding a chimeric immunoglobulin heavy chain.

[0016] It is an object of the present invention to provide a method oftreating an individual exposed to a rabies virus by administering to theindividual a therapeutically effective amount of a human monoclonalrabies virus neutralizing antibody that is encoded in cDNA clonesencoding the antibody heavy and light chains expressed in heterologousexpression systems and purified away from deleterious contaminants,thereby preventing the spread of the rabies virus to the central nervoussystem.

DESCRIPTION OF THE INVENTION

[0017] The present invention provides monoclonal antibodies that bindspecifically to the glycoprotein of various rabies virus strains.Post-exposure treatment with monoclonal antibody, or a mixture of avariety of monoclonal antibodies, will neutralize the rabies virus atthe site of entry and prevent the virus from spreading to the centralnervous system (CNS). Thus, for transdermal or mucosal exposure torabies virus, rabies specific-monoclonal antibodies are instilled intothe bite site, as well as administered systemically. Since viralreplication is restricted almost exclusively to neuronal cells,neutralization and clearance of the virus by the monoclonal antibodiesof the present invention prior to entry into the CNS is an effectivepost-exposure prophylactic.

[0018] Cells

[0019] The human B cells used for hybridization were obtained from theperipheral blood of 5 donors between 7 and 21 days after the third doseof a primary rabies vaccination and 5 rabies-immune donors 10 to 21 daysfollowing administration of booster vaccine. In all cases the vaccineemployed was Rabivac™ human diploid cell vaccine (virus strain PitmanMoore 1503-3M, Behringwerke, Marburg, FRG). All of the donors werenegative in tests for HIV and hepatitis B. The mouse-human hybridheteromyeloma SHM-D33 cells utilized as hybridoma fusion partners (Teng,N. N. et al, Proc. Natl. Acad. Sci. USA 80, 7308, 1983) and B95-8Epstein-Barr Virus (EBV)-transformed marmoset leukocytes used as asource of EBV (Henderson et al., J. Exp. Med. Vol. 76, p. 152, 1977)were obtained from ATCC (Rockville, Md.).

[0020] Rabies Viruses

[0021] To assess the capacity of antibody preparations to neutralize avariety of rabies virus strains, a number of antigenically distinctfixed, laboratory strains, as well as two representative street rabiesviruses, were used. Evelyn-Rokitnicki-Abelseth (ERA), challenge virusstandard, either mouse brain adapted (CVS-24) or cell culture adapted(CVS-11), and Pitman-Moore (PM) fixed strains were obtained from theThomas Jefferson University virus collection. Silver-haired bat rabiesvirus (SHBRV), which has been associated with most of the recent rabiescases in the United States of America, and coyote street rabiesvirus/Mexican dog rabies virus (COSRV), which is a member of the dograbies viruses, were obtained as described (Morimoto et al., Proc.Natl.Acad. Sci. USA, Vol 93, p. 5653, 1996). Virus purification andpreparation of glycoprotein (G) and nucleoprotein (N) have beendescribed elsewhere (Dietzschold et al., World Health Organization,Geneva, p. 175, 1996).

[0022] EBV-Transformation of Human PBLs

[0023] Peripheral blood mononuclear cells (PBMCs) were isolated fromwhole blood by density centrifugation on Ficoll-Paque (AmershamPharmacia Biotech, Piscataway N.J.) as detailed elsewhere (Plebanski etal., Immunology Vol. 75, p. 86, 1992). T cells were then depleted bynegative selection using monoclonal anti-CD2 antibody-coated magneticbeads (Dynal Inc., Lake Success N.Y.) and a magnetic particleconcentrator (Dynal). CD-2-negative cells, primarily B cells, werecollected and immortalized as previously described (Swaminathan, 1992).Briefly, B95-8 cells, cultured to confluency in RPMI₁₆₄₀ (Gibco BRL LifeTechnologies, Grand Island N.Y.) supplemented with 10% fetal bovineserum (FBS; Gibco), were lysed by freeze-thawing on dry ice to releaseintracellular EBV. Supernatant containing EBV was clarified by spinningat 1000 RPM for 10 min and by filtration through a 0.45 μm filter. Viruswas concentrated by centrifugation at 8000 RPM for 2 h at 4° C. 7×10⁶ Bcells (suspended in 1 ml of B95-8 culture media) were incubated at 37°C. for 2 h with virus prepared from 25 mls of B95-8 cells. Followinginfection, the cells were washed twice with culture media, plated in 96well flat-bottom microtiter plates (Nunc, Fisher Scientific, PittsburghPa.) at a concentration of 1×10⁴ cells/well, and cultured at 37° C. in ahumidified atmosphere of 5% CO₂ and 95% air.

[0024] Establishment of Mouse-Human Heterohybrids

[0025] After the EBV-transformed cell lines had been cultured forapproximately 4 weeks, supernatant was harvested and tested for thepresence of rabies virus-specific antibody in ELISA. Positive wells weretransferred first to 1 ml and then to 2 ml cultures (48 and 24 wellplates, Nunc) and the supernatant then assayed in the rapid fluorescentfocus inhibition test (RFFIT) for rabies virus neutralizing antibody, asdetailed elsewhere (Hooper, ASM Press, WA p. 755, 1997). Cell linesproducing neutralizing antibody were hybridized with SHM-D33 cells (ATCCAccession Number CRL1668) as follows. Equal numbers of SHM-D33 andEBV-transformed cells (approximately 5×10⁶ each) were added togetherinto a sterile polystyrene round-bottom tube (Falcon, Fisher Scientific)and centrifuged at 1000 RPM for 10 min. Cells were washed twice withserum-free medium and the cell pellet resuspended in 100 μl of medium.

[0026] Tubes were warmed in a 37° C. water bath for 1 min and then 0.5ml of warm (37° C.) 50% (wt/vol) polyethylene glycol (Sigma ChemicalCo., St. Louis Mo., cat. #P-7181) was added, dropwise over a 45-secperiod while gently shaking the tube. The fusion reaction was thenstopped by the slow addition of 3 ml of serum-free medium over 30 secfollowed by the addition of 9 ml over 30 sec. The tubes were allowed tostand at room temperature for 8 min and then incubated for 2 min in a37° C. water bath. The cells were then centrifuged at 500 g for 3 minand the cell pellet gently resuspended in 30 ml of Iscove's modificationof Dulbecco's (IMDM; Gibco) medium containing 10% FBS, as well as 0.04μM aminopterin (Gibco) and 10 μM oubain (Sigma) to select against cellswhich had not hybridized. Cell suspensions were plated in 96 wellflat-bottom microtiter plates at a concentration of 1×10⁴ cells per welland incubated as described for the lines.

[0027] When colonies of heterohybrid cells had become established(approximately 6 weeks of culture) supernatants were tested for rabiesvirus-specific antibody production in ELISA and RFFIT.Antibody-producing cells were cloned a minimum of three times bylimiting dilution in microtiter plates. Cells were titrated in 96 wellround bottom plates in 2-fold dilutions starting from 4 cells per well.Cells from wells containing an average of 0.25 cells or less wereexpanded for the collection of supernatant and further analysis.

[0028] Analysis of Rabies Virus-Specific Antibodies in ELISA

[0029] Antibody specificity and isotype was assessed in solid phaseELISA. Plates (PolySorb™, Nunc) were coated at room temperature in ahumidified chamber overnight with 5·g/ml rabies ERA virus, glycoprotein,or nucleoprotein diluted in phosphate-buffered saline (PBS). The plateswere then blocked with 5% powdered milk in PBS and washed in PBScontaining 0.05% Tween₂₀ (PBS-Tween) prior to the addition ofsupernatant samples.

[0030] Following incubation at room temperature for 2 h, the plates werewashed with PBS-Tween to remove unbound primary antibody and variousenzyme-conjugated or biotinylated secondary antibodies specific for thevarious human heavy chain isotypes were added for 1 h at roomtemperature. Secondary antibody was detected either by the production ofa soluble end product in the medium upon addition of the appropriatesubstrate (3,3′,5,5′-tetramethylbenzidine (TMB) in phosphate-citratebuffer, or p-nitrophenyl phosphate (PNPP) in 0.1M glycine buffer, Sigma)or following the addition of avidin-alkaline phosphatase (30 min at RT)and PNPP substrate. The peroxidase-TMB reaction was stopped by theaddition of 2M H₂SO₄. Absorbance values were read in a microplatespectrophotometer (Biotek, Winooski Vt.) at 450 nm for the TMB productand at 405 nm for the PNPP reaction.

[0031] RFFIT

[0032] Supernatant samples from each transformed cell line were assayedfor the presence of rabies virus-neutralizing antibodies using avariation of the rapid fluorescent focus inhibition test (RFFIT) aspreviously described (Hooper, ASM Press, WA p. 755, 1997). Supernatantsamples (50 μl) were diluted in 96 well flat-bottom plates (Nunc). 30 μlof a rabies virus dilution known to cause 80-90% infection of theindicator cells were added to each test sample, and the plates incubatedat 37° C. for 1 h. Negative media and positive rabies-immune serumcontrol samples were included in each assay. After incubation, 30 μl ofa 1.8×10⁶ cells/ml concentration of baby hamster kidney (BHK) cells wereadded to each well and the cultures incubated overnight at 37° C. Theplates were then washed once with ice-cold PBS and fixed with ice-cold90% acetone for 20 min at −20° C. After fixation, acetone was removedand the plates were air dried. To detect infected BHK cells, 40 μl ofFITC anti-rabies nucleoprotein monoclonal globulin (Centocor, MalvernPa.) were added to each well for 45 min at 37° C. The plates were thenwashed three times with distilled water and examined under a fluorescentmicroscope.

[0033] Purification of Antibodies by Affinity Chromatography

[0034] IgG1 antibody was purified using a protein A column (rProtein ASepharose™ Fast Flow, Amersham Pharmacia Biotech). Briefly, supernatantswere clarified by filtration through a 0.45 μm membrane and the pHadjusted to 8.0 with 1N NaOH. Supernatant was run through the column ata linear flow rate of approximately 100 cm/hour. After washing in PBS(pH 8), antibody was eluted from the column using a 0.1M citric acidsolution and then dialyzed against PBS.

[0035] IgG3 antibody was purified using a protein G column (Protein GSepharose™ Fast Flow, Amersham Pharmacia Biotech). IgG3-containingsupernatant was clarified by filtration through a 0.45 μm membrane andthe pH adjusted to 7.0 with 1N NaOH. Supernatant was run through thecolumn at a linear flow rate of approximately 11 cm/hour. After washingwith PBS, antibody was eluted from the column using 0.1M glycine buffer,pH 3.0, and then dialyzed against PBS.

[0036] IgM antibody was purified using mannan binding protein and amodification of a previously described technique (Nevens et al., J.Chromatogr, Vol. 597, p. 247, 1992). Briefly, supernatant containing IgMwas EDTA treated, brought to pH 8.0 with 1M NaOH, filtered and cooled to4° C. Mannan binding protein-agarose (Sigma) was washed in a column at4° C. with binding buffer consisting of 0.1M NaHCO₃/0.5M NaCl, pH 8.3and then the supernatant was added and incubated on the column for 15min at 4° C. The column was then washed with several volumes of bindingbuffer and brought to RT for 1 h. The IgM was eluted from the columnwith binding buffer at RT and dialyzed against PBS.

[0037] Protein concentrations of the dialyzed antibody preparations weredetermined using a protein detection assay (Bio-Rad Laboratories,Hercules Calif.) as follows. 100 μl of sample were added to 5 ml of a ⅕dilution of dye reagent concentrate and incubated at RT for 10 minutes.Negative PBS control and various bovine serum albumin (BSA) proteinstandards were included in each assay. After incubation, samples wereread in a spectrophotometer at 595 nm. Protein concentrations of testsamples were calculated with reference to the absorbance of the BSAstandards. The purity of all antibody preparations was assessed byelectrophoresis in 12.5% polyacrylamide gel under reducing conditions(SDS-PAGE). Purified antibodies showed two major bands on SDS-PAGEcorresponding to isolated heavy and light immunoglobulin chains.

[0038] Generation, Isolation and Sequencing of cDNA Clones

[0039] Total RNA was isolated from JA hybridoma cell by using RNAzol B(Biotecx Laboratories, Houston). Reverse transcriptase reactions wereperformed at 42° C. for 1 hr with avian myeloblastosis virus reversetranscriptase (Promega) and oligo(dT) primer. A portion of the reversetranscriptase products were subjected to polymerase chain reaction (PCR)amplification using heavy chain specific primers: IgG-HF1 primer(5′-ACCATGGAGTTTGGGCTGAG-3′ (SEQ. ID. NO: 5), start codon; underline,accession #Y14737), and IgG-HR2 primer (5′-ACTCATTTACCCGGGGACAG-3′ (SEQ.ID. NO: 6), stop codon; underline, accession #Y14737) or light chainspecific primers: IgG-LF5 primer (5′-AGCATGGAAGCCCCAGCTCA-3′ (SEQ. ID.NO: 7), start codon; underline, accession #M63438), and IgG-LR2 primer(5′-CTCTAACACTCTCCCCTGTTG-3′ (SEQ. ID. NO: 8), stop codon; underline,accession #M63438). Amplification was carried out for 35 cycles ofdenaturation at 94° C. for 60 seconds, annealing at 50° C. for 60seconds, and polymerization at 72° C. for 90 seconds with Taq DNApolymerase (Promega). The PCR products (1.4 kb for heavy chain, 0.7 kbfor light chain) were purified and sequenced by using the AmpliTaq cyclesequencing kit (Perkin-Elmer) with the specific primers. The PCRproducts were cloned into TA cloning vector, pCR2.1 (Invitrogen). Thecloned heavy chain and light chain cDNA was sequenced by using theAmpliTaq cycle sequencing kit (Perkin-Elmer) with the specific primers.

[0040] Monoclonal Rabies Virus Neutralizing Antibody Coding Sequences

[0041] Monoclonal antibody cDNA, and sequences complementary thereto,are monoclonal antibody nucleic acids provided by the present invention.In a specific embodiment herein, a monoclonal antibody cDNA sequence isprovided for the heavy chain (SEQ. ID. NO: 1) and the light chain (SEQ.ID. NO: 2) of the monoclonal antibody from clone JA, thus lacking anyintrons.

[0042] The invention also provides single-stranded oligonucleotides foruse as primers in PCR that amplify a monoclonal antibodysequence-containing fragment, for example the variable or hypervariableregion of the monoclonal antibody. The oligonucleotide having thesequence of a hybridizable portion, at least 8 nucleotides, of amonoclonal antibody gene, and another oligonucleotide having the reversecomplement of a downstream sequence in the same strand of the monoclonalantibody gene, such that each oligonucleotide primes synthesis in adirection toward the other. The oligonucleotides are preferably in therange of 10-35 nucleotides in length.

[0043] The present invention provides the full-length cDNA sequences forthe heavy and light chains of the monoclonal antibody of heterohybridomaclone JA (SEQ ID NO: 1 and SEQ ID NO: 2, respectively), and the encodedpolypeptides of #1-474 amino acids for the heavy chain (SEQ ID NO: 3)and #1-234 amino acids for the light chain (SEQ. ID. NO:4).

[0044] In a specific embodiment disclosed herein, the invention relatesto the nucleic acid sequence of the monoclonal antibody fromheterohybridoma clone JA. In a preferred, but not limiting, aspect ofthe invention; the heterohybridoma clone JA is the source of themonoclonal antibody cDNA.

[0045] Functional Equivalents of Monoclonal Rabies Virus NeutralizingAntibodies

[0046] The invention also includes functional equivalents of theantibodies described in this specification. Functional equivalents havebinding characteristics comparable to those of the antibodies, andinclude, for example, chimerized and single chain antibodies, as well asfragments thereof. Methods of producing such functional equivalents aredisclosed in PCT Application WO 93/21319, European Patent ApplicationNo. 239,400; PCT Application WO 89/09622; European Patent Application338,745; and European Patent Application EP 332,424.

[0047] Functional equivalents include polypeptides with amino acidsequences substantially the same as the amino acid sequence of thevariable or hypervariable regions of the antibodies of the presentinvention. “Substantially the same” amino acid sequence is definedherein as a sequence with at least 70%, preferably at least about 80%,and more preferably at least about 90% homology to another amino acidsequence, as determined by the FASTA search method in accordance withPearson and Lipman, Proc. Natl. Inst. Acad. Sci. USA 85, 2444-2448,1988. Chimerized antibodies have constant regions derived substantiallyor exclusively from human antibody constant regions and variable regionsderived substantially or exclusively from the sequence of the variableregion of a monoclonal antibody from each stable heterohybridoma(Champion, J. M., et al., Journal of Immunological Methods, 235 81-90,2000).

[0048] Single chain antibodies or Fv fragments are polypeptides thatconsist of the variable region of the heavy chain of the antibody linkedto the variable region of the light chain, with or without aninterconnecting linker. Thus, the Fv comprises the entire antibodycombining site.

[0049] Functional equivalents further include fragments of antibodiesthat have the same, or substantially the same, binding characteristicsto those of the whole antibody. Such fragments may contain one or bothFab fragments or the F(ab′).sub.2 fragment. Preferably the antibodyfragments contain all six complement determining regions of the wholeantibody, although fragments containing fewer than all of such regions,such as three, four or five complement determining regions, are alsofunctional. The functional equivalents are members of the IgGimmunolglobulin class and subclasses thereof, but may be or may combineany one of the following immunoglobulin classes: IgM, IgA, IgD, or IgE,and subclasses thereof. Heavy chains of various subclasses, such as theIgG subclasses, are responsible for different effector functions andthus, by choosing the desired heavy chain constant region, chimericantibodies with desired effector function are produced. Preferredconstant regions are gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4).The light chain constant region can be of the kappa or lambda type.

[0050] The immunoglobulins of the present invention can be monovalent,divalent or polyvalent. Monovalent immunoglobulins are dimers (HL)formed of a chimeric heavy chain associated through disulfide bridgeswith a chimeric light chain. Divalent immunoglobulins are tetramers(H.sub.2 L.sub.2) formed of two dimers associated through at least onedisulfide bridge.

[0051] Standard Recombinant DNA Techniques

[0052] Standard recombinant DNA techniques are described in Sambrook etal., “Molecular Cloning,” Second Edition, Cold Spring Harbor LaboratoryPress (1987) and by Ausubel et al.(Eds) “Current Protocols in MolecularBiology,” Green Publishing Associates/Wiley-Interscience, New York(1990).

[0053] Briefly, a suitable source of cells containing nucleic acidmolecules that express the desired DNA, such as an antibody or antibodyequivalent, is selected. Total RNA is prepared by standard proceduresfrom a suitable source. The total RNA is used to direct cDNA synthesis.Standard methods for isolating RNA and synthesizing cDNA are provided instandard manuals of molecular biology such as, for example, thosedescribed above.

[0054] The cDNA may be amplified by known methods. For example, the cDNAmay be used as a template for amplification by polymerase chain reaction(PCR); see Saiki et al., Science, 239, 487, 1988 or Mullis et al., U.S.Pat. No. 4,683,195. The sequences of the oligonucleotide primers for thePCR amplification are derived from the known sequence to be amplified.The oligonucleotides are synthesized by methods known in the art.Suitable methods include those described by Caruthers in Science 230,281-285, 1985.

[0055] A mixture of upstream and downstream oligonucleotides are used inthe PCR amplification. The conditions are optimized for each particularprimer pair according to standard procedures. The PCR product isanalyzed, for example, by electrophoresis for cDNA having the correctsize, corresponding to the sequence between the primers.

[0056] Alternatively, the coding region may be amplified in two or moreoverlapping fragments. The overlapping fragments are designed to includea restriction site permitting the assembly of the intact cDNA from thefragments.

[0057] In order to isolate the entire protein-coding regions for theheavy and light chains of each monoclonal antibody from eachheterohybridoma cell line, for example, the upstream PCR oligonucleotideprimer is complementary to the sequence at the 5′ end, encompassing theATG start codon and at least 5-10 nucleotides upstream of the startcodon. The downstream PCR oligonucleotide primer is complementary to thesequence at the 3′ end of the desired DNA sequence. The desired cDNAsequence encodes the entire portion of the heavy and light chains ofeach monoclonal antibody, including the stop codon.

[0058] The cDNA to be amplified, such as that encoding antibodies orantibody equivalents, may also be replicated in a wide variety ofcloning vectors in a wide variety of host cells. The host cell may beprokaryotic or eukaryotic.

[0059] The vector into which the monoclonal antibody cDNA is spliced maycomprise segments of chromosomal, non-chromosomal and synthetic DNAsequences. Some suitable prokaryotic cloning vectors include, but arenot limited to, plasmids from E. coli, such as colE1, pCR1, pBR322,pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include, but are notlimited to, derivatives of phage DNA such as M13 and other filamentoussingle-stranded DNA phages.

[0060] The vector containing the monoclonal antibody cDNA to beexpressed is transfected into a suitable host cell, as described infra.The host cell is maintained in an appropriate culture medium, andsubjected to conditions under which the cells and the vector replicate.

[0061] Chimeric Antibodies

[0062] In general, the chimeric antibodies are produced by preparing,for each of the light and heavy chain components of the chimericimmunoglobulin, a fused gene comprising a first DNA segment that encodesat least the functional portion of the human rabies virus specificneutralizing, preferably glycoprotein, human variable region linked(e.g., functionally rearranged variable region with joining segment) toa second DNA segment encoding at least a part of a human constantregion. Each fused gene is assembled in or inserted into an expressionvector. Recipient cells capable of expressing the gene products are thentransfected with the genes. The transfected recipient cells are culturedunder conditions that permit expression of the incorporated genes andthe expressed immunoglobulins or immunoglobulin chains are recovered.

[0063] Genes encoding the variable region of immunoglobulin heavy andlight chains are obtained from lymphoid cells that produce themonoclonal rabies virus neutralizing antibodies. For example, theheterohybridoma cell lines that produce monoclonal antibody against therabies glycoprotein provide a source of immunoglobulin variable regionfor the present chimeric antibodies. Constant regions are obtained fromhuman antibody-producing cells by standard cloning techniques.Alternatively, because genes representing the two classes of lightchains and the five classes of heavy chains have been cloned, constantregions of human origin are readily available from these clones.Chimeric antibody binding fragments such as F(ab′).sub.2 and Fabfragments are prepared by designing a chimeric heavy chain gene intruncated form. For example, a chimeric gene encoding a F(ab′).sub.2heavy chain portion would include DNA sequences encoding the CH.sub.1domain and hinge region of the heavy chain. Alternatively, suchfragments can be obtained by enzymatic cleavage of a chimericimmunoglobulin. For instance, papain or pepsin cleavage can generate Fabor F(ab′).sub.2 fragments, respectively.

[0064] Preferably, the fused genes encoding the heavy and light chimericchains, or portions thereof, are assembled in two different expressionvectors that can be used to cotransfect a recipient cell. Each vectorcontains two selectable genes, one for selection in a bacterial systemand one for selection in a eukaryotic system, each vector having adifferent pair of genes. These vectors allow production andamplification of the fused genes in bacterial systems, and subsequentcotransfection of eukaryotic cells and selection of the cotransfectedcells. Examples of selectable genes for the bacterial system include,but are not limited to, the genes that confer ampicillin resistance andthe gene that confers chloramphenicol resistance. Two selectable genesfor selection of eukarytoic transfectants are preferred, but are notlimited to: (i) the xanthine-guanine phosphoribosyltransferase gene(gpt), and (ii) the phosphotransferase gene from Tn5 (designated neo).Selection with gpt is based on the ability of the enzyme encoded by thisgene to use xanthine as a substrate for purine nucleotide synthesis; theanalogous endogenous enzyme cannot. In a medium containing xanthine andmycophenolic acid, which blocks the conversion of inosine monophosphateto xanthine monophosphate, only cells expressing the gpt gene cansurvive. The product of the neo blocks the inhibition of proteinsynthesis in eukarytoic cells caused by the antibiotic G418 and otherantibiotics of its class. The two selection procedures can be usedsimultaneously or sequentially to select for the expression ofimmunoglobulin chain genes introduced on two different DNA vectors intoa eukaryotic cell.

[0065] Expression Systems

[0066] Due to the inherent degeneracy of the genetic code, other DNAsequences which encode substantially the same or a functionallyequivalent heavy and light chain amino acid sequences, is within thescope of the invention. Altered DNA sequences which may be used inaccordance with the invention include deletions, additions orsubstitutions of different nucleotide residues resulting in a sequencethat encodes the same, or a functionally equivalent, gene product. Thegene product itself may contain deletions, additions or substitutions ofamino acid residues within a heavy or light chain sequence which resultin a silent change, thus producing a functionally equivalent monoclonalantibody.

[0067] In accordance with the present invention, nucleotide sequencescoding for heavy and light chains of the monoclonal rabies virusneutralizing antibody, a fragment or analog thereof, are inserted intoan appropriate expression vector. This vector which contains thenecessary elements for transcription and translation of the insertedprotein-coding sequence so as to generate recombinant DNA molecules thatdirect the expression of heavy and light chain immunoglobulins for theformation of monoclonal rabies virus neutralizing antibody.

[0068] The preferred recipient cell line is a myeloma cell. Myelomacells can synthesize, assemble and secrete immunoglobulins encoded bytransfected immunoglobulin genes. Further, they possess the mechanismfor glycosylation of the immunoglobulin. A particularly preferredrecipient cell is a myeloma cell line that does not produceimmunoglobulin, such as Sp2/0. These cell lines produce only theimmunoglobulin encoded by the transfected immunoglobulin genes. Myelomacells can be grown in culture or in the peritoneum of mice wheresecreted immunoglobulin can be obtained from ascites fluid. Otherlymphoid cells such as B lymphocytes or hybridoma cells can serve assuitable recipient cells.

[0069] Several methods exist for transfecting lymphoid cells withvectors containing immunoglobulin encoding genes. A preferred way ofintroducing DNA into lymphoid cells is by electroporation. In thisprocedure recipient cells are subjected to an electric pulse in thepresence of the DNA to be incorporated. Another way to introduce DNA isby protoplast fusion. In this method, lysozyme is used to strip cellwalls from bacteria harboring the recombinant plasmid containing theimmunoglobulin gene. The resulting spheroplasts are fused with myelomacells with polyethylene glycol. After protoplast fusion, thetransfectants are selected and isolated. Another technique that can beused to introduce DNA into many cell types is calcium phosphateprecipitation.

[0070] The immunoglobulin genes can also be expressed in nonlymphoidcells, such as bacteria or yeast. When expressed in bacteria, theimmunoglobulin heavy chains and light chains become part of inclusionbodies. Thus, the chains must be isolated and purified and thenassembled into functional immunoglobulin molecules. Other strategies forexpression in E. coli are available (see e.g., Pluckthun, A.,BioTechnology 9:545-551, 1991; Skerra, A. et al., BioTechnology9:273-278, 1991), including secretion from E. coli as fusion proteinscomprising a signal sequence.

1 8 1 1430 DNA Homo sapien 1 accatggagt ttgggctgag ctggcttttt cttgtggctattttaaaagg tgtccagtgt 60 gaggtgcagc tgttggagtc tgggggaggc ttggtacagcctggggggtc cctgagactc 120 tcctgtgcag cctctggatt cacctttagc aactatgccatgagctgggt ccgccaggct 180 ccagggaagg ggctggagtg ggtctcagct attagtgctagtggtcatag cacatatttg 240 gcagactccg tgaagggccg gttcaccatc tccagagacaattccaagaa cacgctgtat 300 ctgcaaatga acagcctgag agccgaggac acggccgtatattactgtgc gaaagatcga 360 gaggttacta tgatagttgt acttaatgga ggctttgactactggggcca gggaacccgg 420 gtcaccgtct cctccgcctc caccaagggc ccatcggtcttccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctggtcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcggcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc agcagcgtggtgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagcccagcaacac caaggtggac 720 aagagagttg agcccaaatc ttgtgacaaa actcacacatgcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaaaacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacgtgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcataatgccaagac aaagccgcgg 960 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcctcaccgtcct gcaccaggac 1020 tggctgaatg gcaaggagta caagtgcaag gtctccaacaaagccctccc agcccccatc 1080 gagaaaacca tctccaaagc caaagggcag ccccgagaaccacaggtgta caccctgccc 1140 ccatcccggg aggagatgac caagaaccag gtcagcctgacctgcctggt caaaggcttc 1200 tatcccagcg acatcgccgt ggagtgggag agcaatgggcagccggagaa caactacaag 1260 accacgcctc ccgtgctgga ctccgacggc tccttcttcctctatagcaa gctcaccgtg 1320 gacaagagca ggtggcagca ggggaacgtc ttctcatgctccgtgatgca tgaggctctg 1380 cacaaccact acacgcagaa gagcctctcc ctgtccccgggtaaatgagt 1430 2 708 DNA Homo sapien 2 agcatggaag ccccagctca gcttctcttcctcctgctac tctggctccc agataccacc 60 ggagaaattg tgttgacaca gtctccagccaccctgtctt tgtctccagg ggaaagagcc 120 accctcgcct gcagggccag tcagactgctagcaggtact tagcctggta ccaacagaaa 180 cctggccagg ctcccagact cctcatctatgatacatcca acagggccac tggcatccca 240 gccaggttca gtggcagtgg gtctgggacagacttcactc tctccatcag cagcctggag 300 cctgaagatt ttgcagttta ttactgtcagcagcgtttca actggccgtg gacgttcggc 360 caagggacca aggtggaatt caaacgaactgtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaactgcctctgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaaggtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaaggacagcacct acagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacacaaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttcaacaggggag agtgttag 708 3 474 PRT Homo sapien 3 Met Glu Phe Gly Leu SerTrp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu ValGln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asn Tyr Ala MetSer Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser AlaIle Ser Ala Ser Gly His Ser Thr Tyr Leu Ala 65 70 75 80 Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr CysAla Lys Asp Arg Glu Val Thr Met Ile Val Val Leu Asn 115 120 125 Gly GlyPhe Asp Tyr Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ser 130 135 140 AlaSer Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 145 150 155160 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu TyrSer 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly ThrGln Thr 210 215 220 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr LysVal Asp Lys 225 230 235 240 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr HisThr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro SerVal Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn SerThr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp TrpLeu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala LeuPro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 370 375 380 MetThr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyAsn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn HisTyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 4234 PRT Homo sapien 4 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu LeuLeu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln SerPro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ala CysArg Ala Ser Gln Thr 35 40 45 Ala Ser Arg Tyr Leu Ala Trp Tyr Gln Gln LysPro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Asp Thr Ser Asn Arg AlaThr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Ser Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val TyrTyr Cys Gln Gln Arg Phe 100 105 110 Asn Trp Pro Trp Thr Phe Gly Gln GlyThr Lys Val Glu Phe Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val PheIle Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala LysVal Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser GlnGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser LeuSer Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His LysVal Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 ValThr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 5 20 DNA Homo sapien 5accatggagt ttgggctgag 20 6 20 DNA Homo sapien 6 actcatttac ccggggacag 207 20 DNA Homo sapien 7 agcatggaag ccccagctca 20 8 21 DNA Homo sapien 8ctctaacact ctcccctgtt g 21

What is claimed is:
 1. Isolated nucleic acid molecules, comprising aheavy chain and a light chain nucleic acid sequence that encodes a heavychain and a light chain amino acid sequence, wherein said heavy chainand light chain amino acid sequences comprise a monoclonal rabies virusneutralizing antibody that specifically binds to a rabies virus protein.2. The isolated nucleic acid molecules of claim 1, comprising a cDNAsequence of a heavy chain (SEQ. ID. NO: 1) and a cDNA sequence of alight chain (SEQ. ID. NO: 2).
 3. An isolated human monoclonal rabiesvirus neutralizing antibody, wherein said isolated human monoclonalrabies virus neutralizing antibody is derived from cDNA clones encodingthe antibody heavy and light chains expressed in heterologous expressionsystems and purified away from deleterious contaminants.
 4. The isolatedhuman monoclonal rabies virus neutralizing antibody of claim 3,comprising a heavy chain and a light chain, wherein said heavy chaincomprises an amino acid sequence of SEQ. ID. NO:3 and said light chaincomprises an amino acid sequence of SEQ. ID. NO:4.
 5. A fused geneencoding a chimeric immunoglobulin light chain, comprising: a) a firstDNA sequence encoding an immunogloublin light chain variable region of amonoclonal rabies virus neutralizing antibody produced by aheterhybridoma cell line; and b) a second DNA sequence encoding a humanlight chain constant region.
 6. An expression vector, comprising a fusedgene of claim
 5. 7. A host cell, comprising an expression vector ofclaim
 6. 8. A fused gene encoding a chimeric immunoglobulin heavy chain,comprising: a) a first DNA sequence encoding an immunogloublin heavychain variable region of a monoclonal rabies virus neutralizing antibodyproduced by a heterhybridoma cell line; and b) a second DNA sequenceencoding a human heavy chain constant region.
 9. An expression vector,comprising a fused gene of claim
 8. 10. A host cell, comprising anexpression vector of claim
 9. 11. An isolated monoclonal rabies virusneutralizing antibody, comprising a fused gene encoding a chimericimmunoglobulin product of claim 6 and a fused gene encoding a chimericimmunoglobulin product of claim
 9. 12. A method of treating anindividual exposed to a rabies virus, comprising: a) administering tosaid individual a therapeutically effective amount of a human monoclonalrabies virus neutralizing antibody of claim 3; and b) preventing aspread of said rabies virus to a central nervous system.